Project description:The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.
Project description:A metagenomic library was prepared using pCC2FOS vector containing about 3.0 Gbp of community DNA from the microbial assemblage of activated sludge. Screening of a part of the un-amplified library resulted in the finding of 1 unique lipolytic clone capable of hydrolyzing tributyrin, in which an esterase gene was identified. This esterase/lipase gene consists of 834 bp and encodes a polypeptide (designated EstAS) of 277 amino acid residuals with a molecular mass of 31 kDa. Sequence analysis indicated that it showed 33% and 31% amino acid identity to esterase/lipase from Gemmata obscuriglobus UQM 2246 (ZP_02733109) and Yarrowia lipolytica CLIB122 (XP_504639), respectively; and several conserved regions were identified, including the putative active site, HSMGG, a catalytic triad (Ser92, His125 and Asp216) and a LHYFRG conserved motif. The EstAS was overexpressed, purified and shown to hydrolyse p-nitrophenyl (NP) esters of fatty acids with short chain lengths (< or = C8). This EstAS had optimal temperature and pH at 35 degrees C and 9.0, respectively, by hydrolysis of p-NP hexanoate. It also exhibited the same level of stability over wide temperature and pH ranges and in the presence of metal ions or detergents. The high level of stability of esterase EstAS with its unique substrate specificities make itself highly useful for biotechnological applications.
Project description:Metagenome-assembled genomes (MAGs) are microbial genomes reconstructed from metagenomic data and can be assigned to known taxa or lead to uncovering novel ones. MAGs can provide insights into how microbes interact with the environment. Here, we performed genome-resolved metagenomics on sequencing data from four studies using sequencing batch reactors at microcosm (~25 mL) and mesocosm (~4 L) scales inoculated with sludge from full-scale wastewater treatment plants. These studies investigated how microbial communities in such plants respond to two environmental disturbances: the presence of toxic 3-chloroaniline and changes in organic loading rate. We report 839 non-redundant MAGs with at least 50% completeness and 10% contamination (MIMAG medium-quality criteria). From these, 399 are of putative high-quality, while sixty-seven meet the MIMAG high-quality criteria. MAGs in this catalogue represent the microbial communities in sixty-eight laboratory-scale reactors used for the disturbance experiments, and in the full-scale wastewater treatment plant which provided the source sludge. This dataset can aid meta-studies aimed at understanding the responses of microbial communities to disturbances, particularly as ecosystems confront rapid environmental changes.
Project description:A new esterase gene, est6, was discovered in an activated sludge metagenomic library. The 729-bp gene encodes a 242-amino acid protein (designated Est6) with a molecular mass of 26.1 kDa. Est6 shared only a moderate identity to a putative hydrolase with the highest BLASTP analysis score. Most of the closely related proteins are uncharacterized and are predicted from genome sequencing data of microorganisms or metagenomic DNA sequences. The phylogenetic analysis of Est6 showed that the protein was assigned to family VI esterases/lipases. The catalytic triad of Est6 was predicted to be Ser135, Asp188, and His219, with Ser135 in a typically conserved pentapeptide (GFSQG) of family VI members, which was further confirmed by site-directed mutagenesis. The est6 gene was overexpressed successfully in its soluble form in Escherichia coli and then purified to its tag-free form and homogeneity by affinity chromatography. The purified Est6 in pH 8.0 buffer was active as a monomer. The optimal conditions for Est6 activity were at a temperature of 45 °C and pH of 8.0 when using p-nitrophenyl acetate as a substrate. The enzyme was stable over wide temperature and pH ranges, and it exhibited activity in the presence of organic solvents, metal cations, or detergents. Furthermore, the enzyme showed significant regioselectivity in the spectrophotometric analysis. In conclusion, Est6 might have the potential for applications in biotechnological processes.
Project description:The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.
Project description:The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.